Broad anti-pathogen potential of DEAD box RNA helicase eIF4A-targeting rocaglates

Inhibition of eukaryotic initiation factor 4A has been proposed as a strategy to fight pathogens. Rocaglates exhibit the highest specificities among eIF4A inhibitors, but their anti-pathogenic potential has not been comprehensively assessed across eukaryotes. In silico analysis of the substitution patterns of six eIF4A1 aa residues critical to rocaglate binding, uncovered 35 variants. Molecular docking of eIF4A:RNA:rocaglate complexes, and in vitro thermal shift assays with select recombinantly expressed eIF4A variants, revealed that sensitivity correlated with low inferred binding energies and high melting temperature shifts. In vitro testing with silvestrol validated predicted resistance in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. Our analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens with rocaglates. Finally, our findings might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.


Supplemental materials
Table S1 -Summary of pathogens that have been previously tested for their sensitivity to a range of natural and synthetic rocaglates  Figure S1: Chemical structures of the four rocaglates tested in this study. All rocaglates are characterized by a cyclopenta [b]benzofurane skeleton, indicated in red, and three benzyl rings, A, B, and C. Silvestrol, the archetypal natural rocaglate, contains a unique 1,4-dioxane moiety, indicated in blue, that increases the possibility of interactions with aa residues beyond those in the RNA-binding pocket. RocA, the first natural rocaglate to be purified and structurally characterized, exhibits the core structure of natural rocaglates. Synthetic rocaglates zotatifin and CR-1-31-B exhibit nitrile and imido groups, respectively, that modulate the binding characteristics of the molecules to the eIF4A:RNA complex. characteristic of ATP dependent DEAD-box RNA helicases, motifs Ib, II, and III, which are characteristic of eIF4A proteins, and six aa residues critical to rocaglate binding. The aa residues at positions 158, 160, 161, 164, 192, and 195 (black arrows) are involved in the protein's interaction with RNA, and residues 182 and 183 (red arrows), located within the DEAD-box (pink), are involved in the interaction with ATP. Position 163 is the primary determinant of sensitivity to rocaglates (green), followed by aa residue 199 (blue) and four conserved aa residues at positions 158, 159, 192, and 195 (yellow). The sequences shown are representative of eIF4A proteins with aa patterns that have been shown to confer rocaglate sensitivity or resistance, including two novel sequences of the rocaglate-producing plants Aglaia stellatopilosa and A. glabriflora, both reported in this study (Accession numbers ON844099 and ON844100, respectively).  158, 159, 163, 192, 195, and 199 (human numbering). Four primary aa patterns were present in all four groups of eukaryotes, representing 63% of all eIF4As (I). Another three patterns were present in three groups (II) and four were present in two groups (III). The largest proportion of patterns, 71%, was only present in one group of eukaryotes and in most cases with only one representative species (IV). Known natural resistance is restricted to only four patterns, including two patterns-TPLFQM and TPGFQI-unique to members of the plant genus Aglaia sp., so far, the only organism known to biosynthesize rocaglates, and its fungal parasite Ophiocordyceps sp. BRM1, respectively.  Table S5. The size of the circles denotes prevalence of the aa pattern among the eIF4As included in our survey.  substitution [53,55]. Mycale hentscheli is deeply rooted in the animal tree, one of the most recent branches of the Opisthokonta. The protein sequence of its eIF4A has not been determined, and no comprehensive map of eIF4A resistance/sensitivity to PatA exists, but potential resistance to PatA, based solely on the distribution of F163L mutations in eIF4A 9 determined by our analysis, is present in early, non-opisthokonta lineages that were presumably never exposed to PatA over evolutionary timescales, an intriguingly similar scenario to the one we describe for rocaglates. It would be interesting to next sequence eIF4A in Mycale hentscheli to determine whether it also contains resistant substitutions and which. with silvestrol but it seems to play an important role also in the eIF4A-(AG) 5 complex formation.
Based on both mutation and docking studies, we suggest the order of importance of Arg residues in RNA and silvestrol binding to be Arg311 > Arg110 > Arg282. Replacing these arginine key residues by alanine, the eIF4A (19-406) :(AG) 5 complex cannot be formed efficiently or at all. Normally, rocaglates increase eIF4A  :(AG) 5 complex stability by raising the T m by 9.3°C (silvestrol), 8.4°C (CR-1-31-B) and 8.0°C (RocA). The R282A mutant shows also increased complex stability after addition of rocaglates (5,0°C silvestrol, 3,9°C CR-1-31-B and 3,6°C RocA) when compared to the mutant protein alone, albeit to a lower extend (see Table S7). This may indicate that ternary complex formation is still possible but with somewhat reduced stability and is probably due to the weak interactions that Arg282 mediates with both the RNA A6 and the dioxane moiety. Therefore, this residue is important but not essential in the complex formation process. For the R110A mutant, there is virtually no difference in melting temperatures upon addition of the rocaglates (see Table   S7). This suggests that rocaglates are not binding anymore to this mutant. Likely, R110 is involved in the eIF4A  :(AG) 5 complex formation due to strong interaction with the phosphate group of RNA G8 which is not occurring in case of the Ala mutant. Moreover, Arg110 mediates only weak interactions with the dioxane moiety.
The most important residue of the series seems to be R311. When this residue is mutated to Ala, T m is reduced upon addition of the rocaglates in a range between -3.1 and -6.3°C (see Table S7) indicating that the protein is evenly destabilized. Arg311 likely establishes a strong salt bridge (orange circle) with the phosphate groups of RNA A7 (yellow sticks with phosphorus atoms colored orange) and in a strong hydrogen bond with the dioxane moiety of silvestrol. An R311A mutant precludes the formation of a stable eIF4A-RNA complex in the presence of rocaglates (see inset).
Most likely, the equilibrium between complex formation and dissociation shifts toward dissociation because of the loss of the strong salt bridge with the RNA. Under these conditions, rocaglates cannot bind as well because the complex is probably not in the most favorable conformation to allow binding to occur. This is potentially the limiting step of complex formation: only when the RNA binds to eIF4A in the optimal conformation, the inhibitors can clamp and bind to the eIF4A-(AG) 5 complex, otherwise they probably bind more loosely, and they even destabilized the whole system. The behavior of the triple mutant is comparable with the single mutant R311A.
Although no synergistic effect of the three mutated Arg residues is noted, a destabilizing effect with T m reduction ranging from -3.2 °C to -3.6 °C is observed (see Table S7).